DNAs coding for flavone synthesis, methods of using flavone synthase DNAS, and plants, flowers, and vectors containing flavone synthase DNAs

ABSTRACT

A DNA encoding a flavone synthase that synthesizes flavones from flavanones, vectors containing said DNAs, and plants expressing same are disclosed. Methods of producing the protein which synthesizes flavones from flavanones, methods of altering the composition of flavonoids or the amount of flavonoids in a plant, methods of altering flower color and plant photosenstivity, and methods of controlling interactions between plants expressing the protein and microorganisms are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent appliacation Ser. No.09/672,785, filed Sep. 29, 2000, now U.S. Pat. No. 6,596,927, which is acontinuation of International Application Nos. PCT/JP00/00490 andPCT/JP00/04379 filed in WIPO on Jan. 28, 2000 and Jan. 30, 2000,respectively, and claims benefit of Japanese patent applications11-22427 and 11-205229 filed respectively on Jan. 29, 1999 and Jul. 19,1999; the entire contents of which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to the control and utilization ofbiosynthesis of flavones, which have effects on flower color, protectionfrom ultraviolet ray, symbiosis with microorganisms, etc. in plants, bya genetic engineering technique. More specifically, it relates to genesencoding proteins with activity of synthesizing flavones fromflavanones, and to their utilization.

BACKGROUND ART

The abundance of different flower colors is one of the pleasant aspectsof life that enriches human minds and hearts. It is expected to increasefood production to meet future population increase by the means ofaccelerating the growth of plants through symbiosis with microorganisms,or by increasing the number of nitrogen-fixing leguminous bacteria, thusimproving the plant productivity as a result of increasing the contentof nitrogen in the soil. Elimination or reduction of the use ofagricultural chemicals is also desirable to achieve more environmentallyfriendly agriculture, and this requires improvement of the soil by theabove-mentioned biological means, as well as higher resistance of plantsagainst microbial infection. Another desired goal is to obtain plantswith high protective functions against ultraviolet rays as a means ofprotecting the plants from the destruction of the ozone layer.

“Flavonoid” is a general term for a group of compounds with a C6-C3-C6carbon skeleton, and they are widely distributed throughout plant cells.Flavonoids are known to have such functions as attracting insects andother pollinators, protecting plant from ultraviolet rays, andparticipating in interaction with soil microorganisms (BioEssays, 16(1994), Koes at al., p. 123; Trends in Plant Science, 1 (1997), Shirley,B. W., p. 377).

Of flavonoids, flavone plays an important role in interaction of plantswith microorganisms, especially in legumes, where they participate inthe initial steps of the symbiosis with leguminous bacteria (Plant Cell,7 (1995), Dixon and Paiva, p. 1085; Annu. Rev. Phytopathol., 33 (1995),Spaink, p. 345). Flavones in petals play a role in recognition byinsects and act as copigments which form complexes with anthocyanins.(Gendai Kagaku, (May, 1998), Honda and Saito, p. 25; Prog. Chem. Org.Natl. Prod., 52 (1987), Goto, T., p. 114). It is known that when flavoneforms a complex with anthocyanin, the absorption maximum of theanthocyanin shifts toward the longer wavelength, i.e. toward blue.

The biosynthesis pathways for flavonoids have been widely studied (PlantCell, 7 (1995), Holton and Cornish, p. 1071), and the genes for all ofthe enzymes involved in the biosynthesis of anthocyanidin 3-glucosideand flavonol, for example, have been isolated. However, the genesinvolved in the biosynthesis of flavones have not yet been isolated. Theenzymes that synthesize flavones include those belonging to thedioxygenase family that depends on 2-oxoglutaric acid (flavone synthaseI) and monooxygenase enzymes belonging to the cytochrome P450 family(flavone synthase II). These groups of enzymes are completely differentenzymes with no structural homology.

It has been reported that in parsley, 2-oxoglutaric acid-dependentdioxygenase catalyzes a reaction which produces apigenin, a flavone,from naringenin, a flavanone (Z. Naturforsch., 36c (1981), Britsch etal., p. 742; Arch. Biochem. Biophys., 282 (1990), Britsch, p. 152). Theother type, flavone synthase II, is known to exist in snapdragon (Z.Naturforsch., 36c (1981), Stotz and Forkmann, p. 737) and soybean (Z.Naturforsch., 42c (1987), Kochs and Grisebach, p. 343; Planta, 171(1987), Kochs et al., p. 519). A correlation has been recently reportedbetween a gene locus and flavone synthase II activity in the petals ofgerbera (Phytochemistry, 49 (1998), Martens and Forkmann, p. 1953).However, there are no reports that the genes for these flavone synthasesI and II were isolated or that flavone synthase II was highly purified.

The properties of a cytochrome P450 protein, which hadlicodione-synthesizing activity that was induced when cultured cells oflicorice (Glycyrrhiza echinata) were treated with an elicitor, wereinvestigated. The protein is believed to catalyze the hydroxylation of2-position of liquiritigenin which is a 5-deoxyflavanone, followed bynon-enzymatic hemiacetal ring opening to produce licodione (PlantPhysiol., 105 (1994), Otani et al., p. 1427). For cloning of licodionesynthase, a cDNA library was prepared from elicitor-treated Glycyrrhizacultured cells, and 8 gene fragments encoding cytochrome P450 werecloned (Plant Science, 126 (1997), Akashi et al., p. 39).

From these fragments there were obtained two different full-length cDNAsequences, each encoding a cytochrome P450, which had been unknown untilthat time. Specifically, they were CYPGe-3 (cytochrome P450 No.CYP81E1)and CYPGe-5 (cytochrome P450 No.CYP93B1, hereinafter indicated asCYP93B1) (Plant Physiol., 115 (1997), Akashi et al., p. 1288). Byfurther expressing the CYP93B1 cDNA in a system using cultured insectcells, the protein derived from the gene was shown to catalyze thereaction synthesizing licodione from liquiritigenin, a flavanone, and2-hydroxynaringenin from naringenin, also a flavanone.

2-Hydroxynaringenin was converted to apigenin, a flavone, by acidtreatment with 10% hydrochloric acid (room temperature, 2 hours). Also,eriodictyol was converted to luteolin, a flavone, by reactingeriodictyol with microsomes of CYP93B1-expressing yeast followed by acidtreatment. It was therefore demonstrated that the cytochrome P450 geneencodes the function of flavanone 2-hydroxylase activity (FEBS Lett.,431 (1998), Akashi et al., p. 287). Here, production of apigenin fromnaringenin required CYP93B1 as well as another unknown enzyme, so thatit was concluded that a total of two enzymes were necessary.

However, no genes have yet been identified for enzymes with activity ofsynthesizing flavones (such as apigenin) directly from flavanones (suchas naringenin) without acid treatment. Thus, despite the fact thatflavones have numerous functions in plants, no techniques have yet beenreported for controlling their biosynthesis in plants, and improving thebiofunctions in which flavones are involved, such as flower color. Thediscovery of an enzyme which by itself can accomplish synthesis offlavones from flavanones and acquisition of its gene, and introductionof such a gene into plants, would be more practical and industriallyapplicable than the introduction into a plant of genes for two enzymesinvolved in the synthesis of flavones from flavanones.

DISCLOSURE OF THE INVENTION

It is an aim of the present invention to provide flavone synthase genes,preferably flavone synthase II genes, and more preferably genes forflavone synthases with activity of synthesizing flavones directly fromflavanones. The obtained flavone synthase genes may be introduced intoplants and over-expressed to alter flower colors.

Moreover, in the petals of flowers that naturally contain large amountsof flavones, it is expected that controlling expression of the flavonesynthase genes by an antisense method or a cosuppression method can alsoalter flower colors. Also, expression of the flavone synthase genes inthe appropriate organs, in light of the antibacterial activity offlavones and their interaction with soil microorganisms, will result inan increase in the antibacterial properties of plants and improvement inthe nitrogen fixing ability of legumes due to promoted symbiosis withrhizosphere microorganisms, as well as a protective effect againstultraviolet rays and light.

The present invention therefore provides genes encoding proteins thatcan synthesize flavones directly from flavanones. The genes are,specifically, genes encoding flavone synthase II that can synthesizeflavones from flavanones by a single-enzyme reaction (hereinafterreferred to as “flavone synthase II”).

More specifically, the present invention provides genes encoding P450proteins having the amino acid sequences listed as SEQ.ID. No. 2, 4 or 8of the Sequence Listing and possessing activity of synthesizing flavonesfrom flavanones, or genes encoding proteins having amino acid sequencesmodified by additions or deletions of one or more amino acids and/or asubstitution with different amino acids in said amino acid sequence, andpossessing activity of synthesizing flavones from flavanones.

The invention further provides a gene encoding proteins having aminoacid sequences with at least 55% identity with the amino acid sequenceslisted as SEQ.ID. No. 2, 4 or 8 of the Sequence Listing and possessingactivity of synthesizing flavones from flavanones.

The invention still further provides genes encoding proteins possessingactivity of synthesizing flavones from flavanones, and hybridizing withall or a part of the nucleotide sequences listed as SEQ.ID. No. 1, 3 or7 of the Sequence List under the conditions of 5×SSC, 50° C.

The invention still further provides a vector, particularly anexpression vector, containing any one of the aforementioned genes.

The invention still further provides a host transformed with theaforementioned vector.

The invention still further provides a protein encoded by any of theaforementioned genes.

The invention still further provides a process for producing theaforementioned protein which is characterized by culturing or growingthe aforementioned host, and collecting the protein withflavone-synthesizing activity from the host.

The invention still further provides a plant into which any one of theaforementioned genes has been introduced, or progenies of the plant or atissue thereof, such as cut flowers, which exhibit the same properties.

The invention still further provides a method of altering amounts andcompositions of flavonoid using the aforementioned genes; a method ofaltering amounts of flavones using the aforementioned genes; a method ofaltering flower colors using the aforementioned genes; a method ofbluing the color of flowers using the aforementioned genes; a method ofreddening the color of flowers using the aforementioned genes; a methodof modifying the photosensitivity of plants using the aforementionedgenes; and a method of controlling the interaction between plants andmicrobes using the aforementioned genes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chromatogram showing the results of HPLC analysis ofproducts obtained from a substrate, naringenin, using proteins encodedby CYP93B1 and TFNS5.

A and B: Obtained by adding a crude enzyme fraction ofCYP93B1-expressing yeast.

C and D: Obtained by adding a crude enzyme fraction of TFNS5-expressingyeast.

A and C: Direct products obtained by addition of enzyme fraction.

B: Products obtained by acid treatment after reaction of A.

D: Products obtained by acid treatment after reaction of C.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Flavanone 2-hydroxylase encoded by the Glycyrrhiza CYP93B1 gene produces2-hydroxyflavanones from flavanones as the substrates, and the productsare converted to flavones by acid treatment. The present inventorsviewed that it would be possible to obtain a gene encoding a flavonesynthase II, which was an object of the invention, by using theGlycyrrhiza-derived cDNA, CYP93B1 for screening of a cDNA library of,for example, a flower containing a large amount of flavones, to thusobtain cDNA encoding proteins with activity of synthesizing flavonesdirectly from flavanones as substrates.

According to the invention, a cDNA library of snapdragon which containsa large amount of flavones is screened using the Glycyrrhiza-derivedcDNA, CYP93B1 as a probe, to obtain cDNA encoding a novel cytochromeP450 (see Example 1). The snapdragon cDNA, ANFNS2, obtained in thismanner and the Glycyrrhiza CYP93B1 cDNA were then used as a mixed probeto obtain TFNS5, a cDNA encoding a novel cytochrome P450, from a cDNAlibrary of torenia flower petals (see Example 2).

The torenia-derived cDNA was expressed in yeast and reacted withnaringenin, a flavanone, as a substrate which resulted in production notof 2-hydroxynaringenin but rather of the flavone apigenin, without acidtreatment (see Example 3). In other words, this enzyme directly producedflavones from flavanones without acid treatment, and its gene wasconfirmed to be a flavone synthase II which had never been cloned. Theamino acid sequence encoded by the snapdragon-derived ANFNS2 of Example1 exhibited high identity of 77% with the flavone synthase II encoded byTFNS5, and it exhibited the enzyme activity of flavone synthase II(Example 4). In addition, since an amino acid sequence encoded byperilla-derived cDNA also exhibited high identity of 76% and 75% withTFNS5 and ANFNS2, respectively (Example 8), it is speculated that theprotein encoded by this cDNA also possesses the same enzymatic activityas the flavone synthases encoded by TFNS5 and ANFNS2.

The genes of the present invention may be, for example, one encoding theamino acid sequences listed as SEQ.ID. No. 2, 4 or 8 of the SequenceListing. However it is known that proteins whose amino acid sequencesare modified by additions or deletions of multiple amino acids and/orsubstitutions with different amino acids can maintain the same enzymeactivity as the original protein. Consequently, proteins having theamino acid sequences listed as SEQ.ID. No. 2, 4 or 8 of the SequenceListing wherein the amino acid sequence is modified by additions ordeletions of one or more amino acids and/or substitutions with differentamino acids, and genes encoding those proteins, are also encompassed bythe present invention so long as they maintain the activity of producingflavones directly from flavanones.

The present invention also relates to genes that have the nucleotidesequences listed as SEQ.ID. Nos. 1, 3 and 7 and nucleotide sequencesencoding the amino acid sequences listed therein, or that hybridize withportions of their nucleotide sequences under conditions of 5×SSC, 50°C., for example, providing they encode proteins possessing activity ofproducing flavones from flavanones. The suitable hybridizationtemperature will differ depending on nucleotide sequences and the lengthof nucleotide sequences, and for example, when the probe used is a DNAfragment comprising 18 bases coding for 6 amino acids, the temperatureis preferably not higher than 50° C.

A gene selected by such hybridization may be a naturally derived one,such as a plant-derived gene, for example, a gene derived fromsnapdragon, torenia or perilla; it may also be a gene from anotherplant, such as gentian, verbena, chrysanthemum, iris, or the like. Agene selected by hybridization may be cDNA or genomic DNA.

The invention also relates to genes encoding proteins that have aminoacid sequences with identity of at least 55%, preferably at least 70%,such as 80% or greater and even 90% or greater, with any one of theamino acid sequences listed as SEQ.ID. Nos. 2, 4 or 8 of the SequenceListing, and that possess activity of synthesizing flavones fromflavanones.

A gene with the natural nucleotide sequence can be obtained by screeningof a cDNA library, for example, as demonstrated in detail in theexamples. DNA encoding enzymes with modified amino acid sequences can besynthesized using common site-directed mutagenesis or a PCR method,using DNA with a natural nucleotide sequence as a starting material. Forexample, a DNA fragment into which a modification is to be introducedmay be obtained by restriction enzyme treatments of natural cDNA orgenomic DNA and then used as a template for site-directed mutagenesis orPCR using a primer having the desired mutation introduced therein, toobtain a DNA fragment having the desired modification introducedtherein. Mutation-introduced DNA fragments may then be linked to a DNAfragment encoding another portion of a target enzyme.

Alternatively, in order to obtain DNA encoding an enzyme consisting of ashortened amino acid sequence, for example, DNA encoding an amino acidsequence which is longer than the aimed amino acid sequence, such as thefull length amino acid sequence, may be cut with desired restrictionendonucleases, and if the DNA fragment obtained thereby does not encodethe entire target amino acid sequence, it may be linked with synthesizedDNA comprising the rest of the sequence.

Thus obtained genes may be expressed in an expression system using E.coli or yeast and its enzyme activity measured to confirm that theobtained gene encodes flavone synthase. By expressing the gene, it isalso possible to obtain the flavone synthase protein as the geneproduct. Alternatively, it is also possible to obtain a flavone synthaseprotein even using antibodies for a full or a partial amino acidsequence listed as SEQ.ID. No. 2, 4 or 8, and such antibodies may beused for cloning of a flavone synthase gene in another organism.

Consequently, the invention also relates to recombinant vectors, andespecially expression vectors, containing the aforementioned genes, andto hosts transformed by these vectors. The hosts used may be prokaryoticor eukaryotic organisms. Examples of prokaryotic organisms that maycommonly be used as hosts include bacteria belonging to the genusEscherichia, such as Escherichia coli, and microorganisms belonging tothe genus Bacillus, such as Bacillus subtilis.

Examples of eukaryotic hosts that may be used include lower eukaryoticorganisms, for example, eukaryotic microorganisms, for example, Eumycotasuch as yeast and filamentous fungi. As yeast there may be mentionedmicroorganisms belonging to the genus Saccharomyces, such asSaccharomyces cerevisiae, and as filamentous fungi there may bementioned microorganisms belonging to the genus Aspergillus, such asAspergillus oryzae and Aspergillus niger and microorganisms belonging tothe genus Penicillium. Animal cells and plant cells may also be used,the animal cells being cell lines from mice, hamsters, monkeys orhumans. Insect cells, such as silkworm cells, or the adult silkwormsthemselves, may also be used.

The expression vectors of the invention will include expressionregulating regions such as a promoter and a terminator, a replicationorigin, etc., depending on the type of hosts into which they are to beintroduced. Examples of promoters for bacterial expression vectors whichmay be used include conventional promoters such as trc promoter, tacpromoter, lac promoter, etc., examples of yeast promoters that may beused include glyceraldehyde-3-phosphate dehydrogenase promoter, PH05promoter, etc., and examples of filamentous fungi promoters that may beused include amylase promoter, trpC, etc. Examples of animal cell hostpromoters that may be used include viral promoters such as SV40 earlypromoter, SV40 late promoter, etc.

The expression vector may be prepared according to a conventional methodusing restriction endonucleases, ligases and the like. Thetransformation of a host with an expression vector may also be carriedout according to conventional methods.

The hosts transformed by the expression vector may be cultured,cultivated or raised, and the target protein may be recovered andpurified from the cultured product, etc. according to conventionalmethods such as filtration, centrifugal separation, cell crushing, gelfiltration chromatography, ion-exchange chromatography and the like.

The present specification throughout discusses flavone synthases IIderived from snapdragon, torenia and perilla that are capable ofsynthesizing flavones directly from flavanones, and it is also knownthat the cytochrome P450 genes constitute a superfamily (DNA and CellBiology, 12 (1993), Nelson et al., p. 1) and that cytochrome P450proteins within the same family have 40% or greater identity in theiramino acid sequences while cytochrome P450 proteins within a subfamilyhave 55% or greater identity in their amino acid sequences, and theirgenes hybridize to each other (Pharmacogenetics, 6 (1996), Nelson etal., p. 1).

For example, a gene for flavonoid 3′,5′-hydroxylase, which was a type ofcytochrome P450 and participated in the pathway of flavonoid synthesis,was first isolated from petunias (Nature, 366 (1993), Holton et al., p.276), and the petunia flavonoid 3′,5′-hydroxylase gene was used as aprobe to easily isolate a flavonoid 3′,5′-hydroxylase gene from gentian(Plant Cell Physiol., 37 (1996), Tanaka et al., p. 711),prairie-gentian, bellflower (WO93/18155 (1993), Kikuchi et al.),lavender, torenia and verbena (Shokubutsu no Kagaku Chosetsu, 33 (1998),Tanaka et al., p. 55).

Thus, a part or all of any of the flavone synthase II genes of theinvention derived from snapdragon, torenia or perilla, which are capableof synthesizing flavones directly from flavanones, can be used as aprobe, in order to obtain flavone synthase II genes capable ofsynthesizing flavones directly from flavanones, from different speciesof plants. Furthermore, by purifying the snapdragon-, torenia- orperilla-derived flavone synthase II enzymes described in thisspecification which can synthesize flavones directly from flavanones,and obtaining antibodies against the enzymes by conventional methods, itis possible to obtain different flavone synthase II proteins that reactwith the antibodies, and obtain genes coding for those proteins.

Consequently, the present invention is not limited merely tosnapdragon-, torenia- or perilla-derived genes for flavone synthases IIcapable of synthesizing flavones directly from flavanones, but furtherrelates to flavone synthases II derived from numerous other plants,which are capable of synthesizing flavones directly from flavanones. Thesources for such flavone synthase II genes may be, in addition tosnapdragon, torenia and perilla described here, also gentian, verbena,chrysanthemum, iris, commelina, centaurea, salvia, nemophila and thelike, although the scope of the invention is not limited to theseplants.

The invention still further relates to plants whose colors are modifiedby introducing a gene or genes for flavone synthases II that cansynthesize flavones directly from flavanones, and to progenies of theplants or their tissues, which may also be in the form of cut flowers.By using the flavone synthases II or their genes which have been clonedaccording to the invention, it is possible to produce flavones in plantspecies or varieties that otherwise produce little or absolutely noflavones. By expressing the flavone synthase II gene or the genes inflower petals, it is possible to increase the amount of flavones in theflower petals, thus allowing the colors of the flowers to be modifiedtoward the blue, for example.

Conversely, by repressing synthesis of flavones in flower petals, it ispossible to modify the colors of the flowers toward the red, forexample. However, flavones have myriad effects on flower colors, and thechanges in flower colors are therefore not limited to those mentionedhere. With the current level of technology, it is possible to introducea gene into a plant and express the gene in a constitutive ortissue-specific manner, while it is also possible to repress theexpression of a target gene by an antisense method or a cosuppressionmethod.

As examples of transformable plants there may be mentioned rose,chrysanthemum, carnation, snapdragon, cyclamen, orchid, prairie-gentian,freesia, gerbera, gladiolus, baby's breath, kalanchoe, lily,pelargonium, geranium, petunia, torenia, tulip, rice, barley, wheat,rapeseed, potato, tomato, poplar, banana, eucalyptus, sweet potato,soybean, alfalfa, lupin, corn, etc., but there is no limitation tothese.

Because flavones have various physiological activities as explainedabove, they can impart new physiological activity or economic value toplants. For example, by expressing the gene to produce flavones inroots, it is possible to promote growth of microorganisms that arebeneficial for the plant, and thus promote growth of the plant. It isalso possible to synthesize flavones that exhibit physiological activityin humans, animals or insects.

EXAMPLES

The invention will now be explained in further detail by way of thefollowing examples. Unless otherwise specified, the molecular biologicalmethods were carried out according to Molecular Cloning (Sambrook etal., 1989).

Example 1 Cloning of Snapdragon Flavone Synthase II Gene

RNA was extracted from about 5 g of young buds of a Yellow Butterflysnapdragon (commercial name by Sakata-no-Tane, KK.), and polyA+ RNA wasobtained by an Oligotex. This polyA+ RNA was used as a template toprepare a cDNA library using a Lambda ZAPII cDNA Library Synthesis Kit(Stratagene) by the method recommended by Stratagene (StratageneInstruction Manual, Revision #065001). The cDNA library was screenedusing the full length cDNA CYP93B1 as the probe. The screening anddetection of positive clones were carried out using a DIG-DNA-labelingand detection kit (Boehringer) based on the method recommended by thesame company, under a low stringent condition.

Specifically, a hybridization buffer (5×SSC, 30% formamide, 50 mM sodiumphosphate buffer (pH 7.0), 1% SDS, 2% blocking reagent (Boehringer),0.1% lauroylsarcosine, 80 μg/ml salmon sperm DNA) was used forprehybridization at 42° C. for 2 hours, after which the DIG-labeledprobe was added and the mixture was kept overnight. The membrane wasrinsed in 5×SSC rinsing solution containing 1% SDS at 65° C. for 1.5hours. One positive clone was obtained, and it was designated as ANFNS1.Upon determining the nucleotide sequence at the 5′ end of ANFNS1 it wasexpected that ANFNS1 encodes a sequence with high identity with theflavanone 2-hydroxylase encoded by licorice CYP93B1, and it was assumedthat it encoded a P450 with a function similar to that of flavanone2-hydroxylase.

However, a comparison with the amino acid sequence of flavanone2-hydroxylase encoded by CYP93B1 suggested that the cDNA of ANFNS1 isnot a full-length cDNA, lacking the portion corresponding toapproximately 65 amino acid residues from the initiating methionine. TheANFNS1 cDNA was therefore used as a probe for-rescreening of thesnapdragon cDNA library, to obtain cDNA (ANFNS2) which was believed toinclude the full-length amino acid sequence. The protein encoded byANFNS2 obtained here exhibited 53% identity on the amino acid level withflavanone 2-hydroxylase encoded by snapdragon-CYP93B1. The nucleotidesequence of ANFNS2 is listed as SEQ.ID. No.1, and the amino acidsequence deduced therefrom is listed as SEQ.ID. No.2.

Example 2 Cloning of Torenia Flavone Synthase II Gene

RNA was extracted from approximately 2 g of buds of a torenia variety(variety name: Sunrenive, Variety Registration Application No.: 7433according to the Seeds and Seedlings Law, by Suntory Ltd.) and thepolyA+ RNA was obtained with an Oligotex. The polyA+ RNA was used as atemplate to prepare a cDNA library using a Lambda ZAPII cDNA LibrarySynthesis Kit (Stratagene) by the method recommended by Stratagene asmentioned in Example 1. The cDNA library was screened using a mixture ofthe aforementioned CYP93B1 cDNA and ANFNS1 cDNA as the probes. Thescreening and detection of positive clones were carried out under thelow stringent conditions as described in Example 1.

One positive clone was obtained, and was designated as TFNS5. Upondetermining the full nucleotide sequence of TFNS5 cDNA, it was foundthat the protein encoded by TFNS5 cDNA exhibited 52% identity on theamino acid level with flavanone-2-hydroxylase encoded by snapdragonCYP93B1. This TFNS5 cDNA also had high identity of 77% with the proteinencoded by ANFNS2, the snapdragon-derived cDNA obtained in Example 1.The determined nucleotide sequence is listed as SEQ.ID. No.3, and theamino acid sequence deduced therefrom is listed as SEQ.ID. No.4.

Example 3 Expression of Torenia Flavone Synthase II Gene in Yeast

The following experiment was conducted in order to detect the enzymeactivity of the protein encoded by TFNS5, the torenia cDNA obtained inExample 2. Parts of the outside of the translated region of the genewere modified to introduce restriction enzyme sites therein to prepare asense primer (5′-AAATAGGATCCAAGCatgGACACAGTCTTAA-3′; underline=BamHIsite; lowercase letters: initiation codon) (SEQ.ID. No.5) and anantisense primer (5′-CCCTTCTAGAtcaAGCACCCGATATTGTGGCCGGG-3′;underline=XbaI site; lowercase letters: termination codon) (SEQ.ID.No.6) were used with KOD polymerase (Toyobo) for PCR reaction. The PCRconditions were 98° C. for one minute, 20 cycles of (98° C. for 15seconds, 55° C. for 10 seconds, 74° C. for 30 seconds), followed by 74°C. for 10 minutes.

After introducing the resultant PCR product into the EcoRV sitepBluescriptII SK(−) (Stratagene), it was digested with restrictionenzymes BamHI and XbaI and introduced at the BamHI-XbaI sites of theyeast expression vector pYES2 (Invitrogen). The resultant plasmid wasthen introduced into BJ2168 yeast (Nihon Gene). The enzyme activity wasmeasured by the method described by Akashi et al. (FEBS Lett., 431(1998), Akashi et al., p. 287). The transformed yeast cells werecultured in 20 ml of selective medium (6.7 mg/ml amino acid-free yeastnitrogen base (Difco), 20 mg/ml glucose, 30 μg/ml leucine, 20 μg/mltryptophan and 5 mg/ml casamino acid), at 30° C. for 24 hours.

After harvesting the yeast cells with centrifugation, the harvestedyeast cells were cultured at 30° C. for 48 hours in an expressing medium(10 mg/ml yeast extract, 10 mg/ml peptone, 2 μg/ml hemin, 20 mg/mlgalactose). After collecting the yeast cells, they were washed bysuspending in water and collecting them. Glass beads were used for 10minutes of disrupting, after which the cells were centrifuged at 8000×gfor 10 minutes. The supernatant was further centrifuged at 15,000×g for10 minutes to obtain a crude enzyme fraction.

A mixture of 15 μg of (R,S)-naringenin (dissolved in 30 μl of2-methoxyethanol), 1 ml of crude enzyme solution and 1 mM NADPH (totalreaction mixture volume: 1.05 ml) was reacted at 30° C. for 2 hours.After terminating the reaction by addition of 30 μl of acetic acid, 1 mlof ethyl acetate was added and mixed therewith. After centrifugation,the ethyl acetate layer was dried with an evaporator. The residue wasdissolved in 100 μl of methanol and analyzed by HPLC. The analysis wascarried out according to the method described by Akashi et al. The acidtreatment involved dissolution of the evaporator-dried sample in 150 μlof ethanol containing 10% hydrochloric acid, and stirring for 30minutes. This was diluted with 1.3 ml of water, 800 μl of ethyl acetatewas further added and mixed therewith, and after centrifugation, theethyl acetate layer was recovered. This was then dried, dissolved in 200μl of methanol, and analyzed by HPLC.

The yeast expressing licorice CYP93B1 produced 2-hydroxynaringenin fromnaringenin, but yielded no apigenin (FIG. 1, A). Only upon acidtreatment of the reaction mixture, apigenin was yielded from2-hydroxynaringenin (FIG. 1, B). In contrast, the yeast expressingtorenia TFNS5 yielded apigenin from naringenin without acid treatment ofthe reaction mixture (FIG. 1, C). This demonstrated that TFNS5 encodes aflavone synthase II.

Example 4 Expression of Snapdragon Flavone Synthase II Gene in Yeast

An approximately 1400 bp DNA fragment obtained by digesting ANFNS2 cDNAwith BamHI and SphI, an approximately 350 bp DNA fragment obtained bydigesting the same with SphI and BamHI, and pYES2 digested with BamHIand XhoI were ligated to obtain a plasmid, which was then introducedinto yeast by the same method as described in Example 3. The resultantrecombinant yeast cells were used to measure the flavone synthesisactivity by the same method as in Example 3. The yeast expressing thesnapdragon-derived ANFNS2 produced apigenin without acid treatment, thusdemonstrating that ANFNS2 encodes a flavone synthase II.

Example 5 Construction of Expression Vector in Plants

A plant expression vector was constructed to introduce TFNS5, thetorenia cDNA obtained in Example 2, into plants. After digestingpBE2113-GUS (Plant Cell Physiol., 37 (1996), Mitsuhara et al., p. 49)with SacI, a blunting kit (Takara) was used to blunt the ends, afterwhich a XhoI linker (Toyobo) was inserted. The resulting plasmid wasthen digested with HindIII and EcoRI, and an approximately 3 kb DNAfragment was recovered. The DNA fragment was linked to the HindIII/EcoRIsite of the binary vector pBINPLUS to prepare pBE2113′. Vector pBINPLUSused here was obtained by modifying the binary vector Bin19 (Nucl. AcidsRes., 12 (1984), Bevan, p. 8711), which is widely used for geneintroduction into plants using Agrobacterium cells, in the mannerreported by van Engelen et al. (Transgenic Research, 4 (1995), vanEngelen et al., p. 288).

The TFNS5 cDNA was cut out of SK(−) vector by cleavage with BamHI/XhoI,and an approximately 1.7 kb fragment thus obtained was ligated to theBamHI/XhoI sites of the aforementioned binary vector pBE2113′. Theconstruct thus obtained, pSPB441, expresses TFNS5 cDNA in the sensedirection under the control of 35S cauliflower mosaic virus promoterhaving a double repeat of the enhancer sequence (Plant cell Physiol., 37(1996), Mitsuhara et al., p. 49).

Example 6 Alteration of Torenia Flower Color

A torenia variety (variety name: Sunrenive, Variety RegistrationApplication No.: 7433 according to the Seeds and Seedlings Law, bySuntory Ltd.) was transformed with pSPB441 constructed in Example 5above, according to the method of Aida et al. (Breeding Science, 45(1995), Aida et al., p. 71). Over 95% of the obtained transformantsshowed alteration of the flower color from the dark purple of the parentstrain to a light purple. The left and the right flower petal colors offour flower petals were measured. While the flower petal color of theparent strain was Number 89A according to the Royal HorticulturalSociety Color Chart, the typical flower petal colors of thetransformants were 82C, 87D, 87C, 88D, 91A, etc. These results indicatedthat introduction of TFNS5 into plants can alter flower colors.

In the transformed individuals, the amount of flavones ranged ⅕ to 1/10that of the host, while the amount of anthocyanins was reduced to about⅓ that of the host. Also detected were flavanones (naringenin,eriodictyol and pentahydroxyflavanone) which are flavone biosynthesisprecursors that were not detected in the host.

Example 7 Expression of Flavone Synthase in Petunias

Plasmid pSPB441 was introduced into a petunia variety (variety name:Revolution Violet Mini, Variety Registration Application No.: 9217,according to the Seeds and Seedlings Law, by Suntory Ltd.) according tothe method of Napoli et al. (Plant Cell, 2, (1990), Napoli et al., p.279). Changes in flower colors occurred in two of the resultanttransformants, where the flower colors were lighter than the parentstrain. The flower color of the parent strain was Number 88A accordingto the Royal Horticultural Society Color Chart, whereas 87A in thetransformants. Also, while no flavones were detected in the parentstrain, a flavone, luteolin, was detected in the transformed strains.

Example 8 Cloning of Perilla Flavone Synthase II Gene

The method described in Example 1 was used to screen a cDNA libraryprepared from leaves of red perilla (Perilla frutescens) according tothe method of Gong et al. (Plant Mol. Biol., 35, (1997), Gong et al., p.915) using λgt10 (Stratagene) as the vector. Culturing, DNA preparationand subcloning of the resulting phage clones #3 were carried outaccording to the method of Gong et al. (Plant Mol. Biol., 35, (1997),Gong et al., p. 915), and the nucleotide sequence was determined andlisted as SEQ.ID. No.7. The deduced amino acid sequence encoded by thisnucleotide sequence was listed as SEQ.ID. No.8. This amino acid sequenceshowed 76% and 75% identity with TFNS5 and ANFNS2, respectively. It alsoshowed 52% identity with CYP93B1.

Example 9 Expression of Perilla Flavone Synthase II Gene in Yeast

The phage clone #3 obtained in Example 8 was used as a template for PCRby the method described in Example 3, using Lambda Arm Primer(Stratagene). The amplified DNA fragment was subcloned at the EcoRV siteof pBluescript KS(−). A clone with the initiation codon of perillaflavone synthase II cDNA on the SalI site side of pBluescript KS(−) wasselected, and was designated as pFS3. The nucleotide sequence of thecDNA insert of pFS3 was determined, and PCR was conducted to confirm theabsence of errors.

An approximately 1.8 kb DNA fragment obtained by digesting pFS3 withSalI and XbaI was ligated with pYES2 digested with XhoI and XbaI(Example 3) to obtain a plasmid which was designated as pYFS3, and thiswas introduced into yeast BJ2168 by the method described in Example 3.When the flavone synthase activity of this recombinant yeast wasmeasured by the method described in Example 3, production of apigeninfrom naringenin was confirmed, indicating that the perilla phage clone#3 cDNA encodes a protein with flavone synthase II activity.

INDUSTRIAL APPLICABILITY

It is possible to alter flower colors by linking cDNA of the inventionto an appropriate plant expression vector and introducing it into plantsto express or inhibit expression of flavone synthases. Furthermore, byexpressing the flavone synthase genes not only in petals but also inentire plants or their appropriate organs, it is possible to increasethe resistance agasint microorganisms of plants or to improve thenitrogen fixing ability of legumes by promoting association withrhizosphere microorganisms, as well as to improve the protective effectsof plants against ultraviolet rays and light.

1. An isolated DNA or RNA encoding a protein, wherein said protein hasan activity of synthesizing flavones from flavanones, further whereinthe protein has the amino acid sequence as shown in SEQ ID NO:
 8. 2. Anisolated DNA having the nucleotide sequence of SEQ ID NO:
 7. 3. A vectorcomprising the isolated DNA according to claim
 1. 4. A plant hosttransformed by the vector according to claim
 3. 5. A method of producinga protein with flavone-synthesizing activity, comprising culturing orgrowing a plant host according to claim 4, and recovering said proteinfrom said plant host.
 6. A plant, a progeny thereof, or a tissuethereof, wherein said plant, progeny, or tissue comprises the isolatedDNA or RNA of claim
 1. 7. A cut flower from the plant or the progenythereof according to claim 6 comprising said isolated DNA or RNA.
 8. Amethod of altering a composition of flavonoids or amount thereof, orboth the composition and amount in a plant host comprising: introducinga vector having the DNA according to claim 1 into a plant host;culturing or growing said plant host; and recovering said flavones;wherein said flavones are included in a composition of flavonoids. 9.The method according to claim 8, wherein said DNA is inserted in thevector.
 10. A method of altering the amount of a flavone in a plant hostcomprising: introducing a vector having the DNA according to claim 1into a plant host; and culturing or growing said host, wherein said DNAis expressed and amount of flavone altered.
 11. A method of altering thecolor of a flower comprising: introducing a vector having the DNAaccording to claim 1 into a flower plant; and culturing or growing saidflower plant, wherein said DNA is expressed and expression of said DNAalters the flower color in said flower plant.
 12. A method of bluing thecolor of a flower comprising: introducing a vector having the DNAaccording to claim 1 into a flower plant; and culturing or growing saidflower plant, wherein the DNA is expressed and flavone is produced inthe flower plant.
 13. A method of reddening the color of a flowercomprising: introducing a vector having the DNA according to claim 1into a flower plant; and culturing or growing said flower plant, whereinexpression of the DNA is suppressed in the flower plant.
 14. A method ofaltering the photosensitivity of a plant comprising: introducing avector having the DNA according to claim 1 into a plant; and culturingor growing said plant, wherein the DNA is expressed.
 15. A method ofcontrolling the interaction between a plant and microorganismscomprising: introducing a vector having the DNA according to claim 1into a plants; and culturing or growing said plant, wherein the DNA isexpressed and flavone produced in the plant.
 16. A method of altering acomposition of flavones or amount thereof, or both the composition andamount in a plant or tissue thereof comprising: introducing a vectorhaving the DNA according to claim 1 into a plant or tissue thereof; andculturing or growing said plant or tissue, whereby the is DNA expressedin said plant or tissue.
 17. A method of suppressing flavone synthesisin a plant or tissue thereof, comprising: introducing a vector havingthe DNA according to claim 1 into a plant or tissue thereof; andculturing or growing said plant or tissue, whereby expression of the DNAis suppressed in said plant.
 18. A method of increasing an amount offlavone in a flower petal, comprising: introducing a vector having theDNA according to claim 1 into a plant or tissue thereof; and culturingor growing said plant or tissue, whereby the DNA is expressed in saidplant.
 19. A method of decreasing an amount of flavone in a flowerpetal, comprising: introducing a vector having the DNA according toclaim 1 into a plant or tissue thereof; and culturing or growing saidplant or tissue, whereby expression of the DNA is suppressed in saidplant.
 20. A method of synthesizing a flavone from flavanone comprising:introducing a vector having the DNA according to claim 1 into a planthost having flavanones; culturing or growing said plant host, wherebysaid DNA is expressed in said plant host to produce a flavone synthase;and said flavone synthase synthesizes flavone from said flavanone.
 21. Amethod for producing a flavone synthase comprising: introducing a vectorhaving the DNA according to claim 1 into a plant host; and culturing orgrowing said plant host, whereby said DNA is expressed in said planthost to produce a flavone synthase.